CN109260228A - A kind of compound for treating tumour - Google Patents

Abstract

The invention belongs to biomedicine technical field, a kind of claimed medicinal composition for being used to prepare treatment tumour comprising, ultimate density 300, the BHK-21 fibroblast of 000~625,000/mL；The artificial basement membrane of 30-60% and 2~10% fetal calf serum Dulbecco's culture solution.

Description

A kind of compound for treating tumour

Technical field

Biomedicine technical field of the present invention, and in particular to treat the compound of tumour.

Background technique

It is shown according to newest Cancer in China epidemic data, kidney and pelvic cancer are that the common uropoiesis of China second is raw The malignant tumour of system is grown, every year about 66,800 new cases and 23,400 Lethal cases.Especially clear-cell carcinoma (Renal Cell Carcinoma, RCC), accounts for significant proportion (Chen, Zheng et in adult kidney neopathy rate al.2016).Different from the lower tumour of some grade malignancies, clear-cell carcinoma has height metastatic and refractiveness, and chemistry With radiotherapy to it all without effective therapeutic effect (Merza and Bilusic 2017, Shingarev and Jaimes 2017)。

Currently, most widely used method is chemotherapy, radiotherapy, hand in cancer (including clear-cell carcinoma) treatment Art, hormonotherapy and " targeting " therapy etc., immunotherapy for cancer and monoclonal antibody therapy are most common two in targeted therapy Kind.

There is following concrete analysis to existing anti-cancer technology:

1. chemotherapy: chemotherapy would generally common are nausea and vomiting, alopecia, be immunized with different degrees of side effect Power decline and loss of appetite etc., while the normal cell of chemotherapy on human body also has damage (Munker, Gerken et al.2018；Oun,Moussa et al.2018；Beaver and Magnan 2016).

2. radiotherapy: although its side effect is serious not as good as chemotherapy, the patient for receiving radiotherapy often will appear skin Skin reaction, such as furfur, erythema and fibrosis.In addition, this method also needs expensive equipment and professional environment (for example to cure Institute), and also have the risk (Gieger and Nolan 2017) for inducing new cancer.

3. immunotherapy: although this method achieves infusive effect in the treatment of cancer, it is well known that exempting from Epidemic disease therapy needs to rely on the immune system of people, so will appear a series of symptom of immune responses, such as fever, nausea and vomiting, entirely Body pain also has cardiopulmonary toxicity if serious, and its therapeutic effect can significantly reduce immunotherapy with advancing age (Hurez,Padron et al.2018)。

4. hormonotherapy (Hormone Therapy): the most common typical side effect of hormonotherapy is that " Cushing sample is comprehensive Sign ", it can also be with steroids type ulcer, behavior or (the Lin et such as insanity and the lesion of muscle and bone al.2017)。

From the foregoing, it can be seen that all there are many kinds of defect and deficiencies for the method for existing treating cancer.

Summary of the invention

The problem of being solved of the invention is to provide a kind of compound for treating tumour.

First aspect present invention provides a kind of compound for treating tumour comprising: ultimate density 300,000~625, The BHK-21 fibroblast of 000/mL；The artificial basement membrane of 30-60% and 2~10% fetal calf serum Dulbecco's training Nutrient solution (DMEM).

In currently preferred technical solution, the medicinal composition further includes 2~10% fetal calf serum Dulbecco's culture solution.

In currently preferred technical solution, the fibroblastic ultimate density of BHK-21 is 300,000~625,000/ mL。

In currently preferred technical solution, artificial basement membrane mass percent is 30%-60%.

In currently preferred technical solution, compound includes: that ultimate density is 300, the BHK- of 000~625,000/mL 21 fibroblasts；The artificial basement membrane of 30%-60%, the Dulbecco's culture solution (DMEM) of 5% fetal calf serum.

In currently preferred technical solution, artificial basement membrane isMatrigel.

Second aspect of the present invention provides the purposes of above-mentioned compound, that is, is used to prepare the drug for the treatment of tumour.

In a currently preferred technical solution, the compound is used to prepare the drug for the treatment of kidney.

In currently preferred another technical solution, the compound is used to prepare the drug for the treatment of lung cancer

In currently preferred technical solution, the compound plays a role at a temperature of 23-37 DEG C, preferably in 25- It plays a role at a temperature of 37 DEG C.

The preparation method of compound of the invention: first by the 30-60% of a certain number of BHK-21 cells and certain volume The artificial basement membrane of ice mixes, and prepares resulting mixture, then adds a certain number of cancer cells, makes the concentration of both cells Between 300,000~625,000/mL, and it is inoculated into culture plate until artificial basement membrane coagulates in 24-37 DEG C of temperature Gu finally adding Dulbecco's Modified Eagle Medium (DMEM) the culture solution training of 2~10% fetal calf serums It supports；Above-mentioned cancer cell preferably uses red fluorescent protein (Red Fluorescence Protein；RFP) labeled.

BHK-21: being a kind of fibroblastic cell strain derived from newborn hamster kidney, by Stoker and Macpherson built strain (Stoker and Macpherson, 1961) in 1961 first.Since BHK-21 is non-to virus infection Often sensitive, this cell is widely used in the host of virus breeding and bio-pharmaceuticals production for many years.

Artificial basement membrane is a kind of basement membrane extract of reconstruction, usually mainly as microenvironment in analogue body for external The 3D of cell is cultivated.(BD Bioscience) is initially from the Engelbreth-Holm- for being rich in extracellular matrix protein The soluble 3D culture medium with colloid reticular structure abundant extracted in Swarm (EHS) murine sarcoma.Due to base Containing the high molecular weight protein in a large amount of extracellular matrix in matter glue, such as laminin (60%), collagen (30%), interior Actin (8%) and heparitin sulfate glycoprotein, and similar FGF, EGF, largely stimulation cell and signal pass TGF-β etc. Lead required growth factor, matrigel being capable of the largely structure of analogue body inner cell basilar memebrane, composition, physical characteristic And function, thus to the in vitro culture of cell provide advantageous growth conditions (Hughes, Postovit et al.2010, Benton,Kleinman et al.2011,Benton,Arnaoutova et al.2014)。

It is a kind of new compound of unprecedented treating cancer that the application is to be protected: BHK-21 cell and matrigel are multiple Close (i) formula and its (ii) application that object is used as antitumor and anticancer agent in cancer treatment.This compound is it is verified that can be efficient Ground inhibits the proliferation diffusion of tumour.Compared to the prior art, compound of the invention, has the advantages that

1. different from the expensive radiotherapy of instrument of chemotherapy and needs for usually requiring expensive reagent, compound of the invention has Better cost-effectiveness, estimation do not exceed 2000 RMB generally.

2. traditional chemotherapy is to go to kill cancer cell using cytotoxic compound.In contrast, of the invention The therapeutic effect of compound is controlled and is pressed down by the cancer cell affinity (Fig. 2 shown in) intrinsic to BHK-21 vascular system Make its growth.

3. CaKi-1 cell has the tendency that sticking to BHK-21 vascular system in matrigel.Its actual effect is invention In BHK-21 cell be scattered nigh any cancer cell (Fig. 3 and Fig. 4) can be removed as one " dust catcher ".

The present invention is a kind of compound being made of following two main component, i.e. a kind of (i) entitled matrigelBasement membrane extract (Basement Membrane Extract), be existing commercial product in the market； (ii) baby hamster kidney cell (BHK-21) is a kind of attached cell for being usually used in expressing albumen.

In the present patent application, it illustratesCompound is being directed to kidney cancer cell (CaKi-1) and lung cancer Cell (Lung Adenocarcinoma, A549) is proliferated the double action that proliferation has capture and inhibits.The present invention is not only It can be used as the feasible method of novelty in terms of anti-curing cancers, also believe and also have in terms of intellectual property's exploitation and commercialization There are huge potentiality.

Detailed description of the invention

Fig. 1 is the ideograph of one embodiment of the invention, and Figure 1A is control group, and 1B is experimental group.

Fig. 2A is CaKi-1 cell and individual matrix gel inoculated and cultured, and Fig. 2 B is shown in CaKi-1 under simple microscope Positioned in compound on the vascular of BHK-21 cell.

Fig. 3 is that for CaKi-1 cell adhesion on the vascular of BHK-21 cell, Fig. 3 A is light field background under fluorescence microscope, Fig. 3 B is the CaKi-1 cell that the RFP of fluorescence microscopy microscopic observation is marked, and Fig. 3 C is the above two compound picture, it is shown that red The CaKi-1 cell adherence of fluorescent protein labeling is outside BHK-21 vascular.

Fig. 4 is the procedure chart that compound of the present invention wraps up that CaKi-1 cell forms orbicule.

Fig. 5 A is the CaKi-1 cell controls figure under the CaKi-1 cell and similarity condition individually cultivated with BHK-21 mixed breeding； Fig. 5 B is the histogram of 5A.

The A549 cell of Fig. 6 A red fluorescent protein (RFP) label is mixed and same with individual 50% matrigel Under the conditions of the A549 cell that marks of RFP and compound be mixed together the comparative diagram of culture；Fig. 6 B is at the end of testing in embodiment 4 The A549 cell of survival is counted.It shows compound package A549 cell and inhibits its proliferation growth.

Specific embodiment

The specific embodiment of the invention is described below in conjunction with attached drawing.

One, drug and reagent:

2.1 drugs:(BD Bioscience)；BHK-21 cell strain (Shanghai cell bank GNHa10)；A549 is thin Born of the same parents' strain (Shanghai cell bank TCHu150)；CaKi-1 cell (Shanghai cell bank TCHu135)

2.2:LS-55 Fluorescence Spectrometer (PerkinElmer Life Sciences), C1-Si laser confocal microscope (Nikon), fluorescence microscope (Nikon), inverted fluorescence microscope (Nikon), multi-function microplate reader are just being set (ThermoScientific), II flow cell sorter of FACSAria (BD Bioscience), automatic cell counter (Invitrogen)。

Two, embodiment

(1) Fig. 1 is ideograph of the invention, simply summarises research contents and method of the invention.

In order to establish the experimental model, in the case where there is solid phase support, cancer cell and BHK-21 cell with certain Concentration ratio, which is suspended in culture solution, makees 3D culture.By taking the CaKi-1 cell of research as an example: first importing red fluorescent protein simultaneously Stablize expression in kidney cancer cell CaKi-1, then with CaKi-1 625,000/mL:BHK-21 625,000/mL:Matrigel The concentration ratio of 50%in DMEM mixes.Control group is added to alone in 50% matrigel with the CaKi-1 of identical quantity, Then two groups of cells are planted respectively in culture hole and is placed it in 37 DEG C until it becomes solid-like, finally added and contain In the complete culture solution of 5% fetal calf serum, it is placed into 5%CO2/37 DEG C of incubator and cultivates 37 days.

In the control group of Figure 1A, CaKi-1 cell is individually wrapping in 50% matrigel；In the experimental group of 1B, CaKi-1 cell is inoculated into 50% matrigel and in the compound of 625,000/mL:BHK-21 cell.

(2) embodiment 1

CaKi-1 cell is adsorbed in the blood vessel wall of BHK-21 cell in matrix gel

Method:

1. buying CMV-mCherry-T2A-Luc slow virus and relevant antibiotic (Ji Man Biotechnology Co., Ltd).

2. being inoculated with common CaKi-1 cell on the culture dish of 10cm, cultivated with complete culture solution.Culture completely Liquid are as follows: 5% fetal calf serum, Pen .- Strep solution (1X) and DMEM culture solution.

3., with above-mentioned slow virus infected cell, then being used when CaKi-1 cell density reaches 60%-80% Blasticidin is screened, until obtaining the CaKi-1 cell of RFP label

4. being screened again to the CaKi-1 cell of infection with flow cell sorter, the highly expressed cell of RFP is obtained.

5. being cultivated with complete culture solution BHK-21 cell, until obtaining sufficient amount of cell.

6. the CaKi-1 cell of the complete culture solution culture height expression RFP with the Blasticidin containing 5ug/ml, until foot The cell of enough amounts is with spare.

7. collecting both the above cell respectively with pancreatin, then counted with automatic cell counter.

8. 50,000 CaKi-1 cells are resuspended with the culture solution of 40ul, the cold Matrigel of 40ul is then added, after mixing It is instilled in 24 orifice plates immediately, 5ul is instilled in each hole, is repeated 6 times altogether.

9. after said mixture instills 24 orifice plates, then in 37 DEG C of placement a period of times, until its formation is hemispherical simultaneously Solidification onboard, finally adds complete culture solution and is cultivated.

10. being suspended 50,000 BHK-21 cells with the Matrigel of 40ul ice, it is prepared into compound, is then cultivated with 40ul 50,000 CaKi-1 mixing with cells that liquid suspends, are inoculated into 24 orifice plates in the same way and are cultivated.

11. being observed and being photographed to record with inverted fluorescence microscope.

As a result: such as Fig. 2A, 2B, Fig. 3 A, 3B, shown in 3C

The CaKi-1 cell of 625,000/ml concentration and independent 50% matrix gel inoculated and cultured is shown in Fig. 2A；

Fig. 2 B is CaKi-1 inoculated and cultured together with the compound that same concentration BHK-21 cell and matrigel form；

Fig. 3 A is the form of the BHK-21 cell in compound under the green fluorescence of fluorescence microscope；

Fig. 3 B is the CaKi-1 cell that the RFP of fluorescence microscopy microscopic observation is marked；

Fig. 3 C shows the CaKi-1 cell adherence of red fluorescent protein marker outside BHK-21 vascular.

In the case where lacking BHK-21, for CaKi-1 after 37 DEG C are cultivated 5 days, inoculum forms all size Independent cell group；And in the presence of BHK-21, CaKi-1 cell is largely adhered in the intercellular vascular of BHK-21, Independent cell mass can not be formed

Conclusion: the present embodiment effectively demonstrates inhibition effect that compound of the present invention spreads kidney cancer cell CaKi-1. Attraction and adhesive attraction of the BHK-21 vascular system to CaKi-1 cell in matrix gel, the CaKi-1 cell quilt in matrix gel It is adsorbed onto the blood vessel wall of BHK-21 cell.

Embodiment 2

Method:

1. with 1-7 and 9 steps in embodiment 1.

2. complete culture solution is added and is trained after the CaKi-1 cell and compound of RFP label are solidified on plate It supports.

3. being observed continuously and being photographed to record with inverted microscope.

As a result: if Fig. 4 shows the picture of 0-6 days continuous micro- sem observations, inner cell is formed in matrix gel within 6 days BHK-21/CaKi-1 sphere.

Conclusion: the sphere for being similar to cocoon-like can be collectively formed in CaKi-1 and compound within one week.

Embodiment 3

Method:

1. with 1-11 step in embodiment 1.

2. all cells are cultivated in 50% matrix gel to after 37 days, with trypsase (Trypsin)/EDTA to its into Row processing, until the dissolution of BHK-21/CaKi-1 orbicule and CaKi-1 cell suspend.

3. collecting two groups of all cells respectively, the CaKi-1 cell of wherein red fluorescent protein marker is counted.

4. directly being counted after collection with automatic cell counter for the CaKi-1 cell individually cultivated.

5. all cells of collection are resuspended in the culture of 100ul for the CaKi-1 cell being mixed with compound In liquid, 10ul is taken out, CaKi-1 cell is counted under fluorescence microscope, then calculates the sum of CaKi-1.

It is tested 6. each sample carries out 4 repetitions.

As a result: as Fig. 5 A CaKi-1 cell for showing that red fluorescent protein (RFP) is marked individually is cultivated in 50% matrix 37 days in gel；Under identical condition, the CaKi-1 cell of label and compound are mixed together culture 37 days.It is thin in BHK-21 In the case that born of the same parents are not present, CaKi-1 cell is proliferated rapidly, almost covers the whole surface of culture hole；And it is mixed in two kinds of cells When closing culture, CaKi-1 cell is captured and is enclosed in the sphere of BHK-21 formation, cannot be diffused into the region of surrounding.

Fig. 5 B intuitively shows the CaKi-1 cell individually cultivated after 37 days, and quantity reaches about 495,000, and same batten With the CaKi-1 cell of BHK-21 mixed breeding under part, finally only survive about 850.In short, the CaKi- with compound co-incubation Cell number (p < 0.0001) when 1 cell number is individually cultivated well below it.

Conclusion: the orbicule that compound is formed traps CaKi-1 cell, and CaKi-1 cell is made to be closed in its ball In vivo, the proliferation diffusion of CaKi-1 cell is prevented:

Embodiment 4

Method:

With embodiment 3

As a result: as Fig. 6 A shows that the A549 cell that red fluorescent protein (RFP) is marked and individual 50% matrigel are mixed Close culture 25 days；With under similarity condition, the A549 cell and compound of RFP label are mixed together culture 25 days.In compound When without containing BHK-21 cell, A549 cell fast breeding, and spread to surrounding；And A549 cell is mixed with complete compound When culture, A549 cell is wrapped in the orbicule of compound formation, cannot be spread to peripheral region.

Fig. 6 B is to count at the end of testing to the A549 cell of survival, and the A549 cell cultivated alone is in last cell Quantity proliferation has arrived about 97,200；And it only survives about 152 with the A549 cell of compound mixed culture.So with multiple Close object mixed culture A549 cell number seldom in its individually cultivate when cell number (p < 0.0001).

Conclusion: the orbicule that compound is formed can also trap A549, by A549 cell encapsulation in orbicule, And inhibit its proliferation diffusion.

The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry Personnel only illustrate the present invention it should be appreciated that the present invention is not limited by examples detailed above described in examples detailed above and specification Principle, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these variation and Improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by appended claims and its is equal Object defines.

Claims (10)

1. a kind of compound for treating tumour, which is characterized in that the compound includes: that ultimate density is 300,000~625, The BHK-21 fibroblast of 000/mL；The artificial basement membrane of 30-60%.

2. compound according to claim 1, which is characterized in that the medicinal composition further includes 2~10% tire ox The Dulbecco's culture solution of serum.

3. compound according to claim 1, which is characterized in that the fibroblastic ultimate density of BHK-21 is 300,000~625,000/mL.

4. compound according to claim 1, which is characterized in that the artificial basement membrane mass percent is 30%- 60%.

5. compound according to claim 1, which is characterized in that the compound include: ultimate density be 300,000~ The BHK-21 fibroblast of 625,000/mL；The artificial basement membrane of 30%-60%, the Dulbecco's training of 5% fetal calf serum Nutrient solution.

6. compound according to claim 1, which is characterized in that the artificial basement membrane isMatrigel.

7. the drug that any one of the claim 1-6 compound is used to prepare treatment tumour.

8. purposes according to claim 7, which is characterized in that the compound is used to prepare the drug for the treatment of kidney.

9. purposes according to claim 7, which is characterized in that the compound is used to prepare the drug for the treatment of lung cancer.

10. purposes according to claim 7, which is characterized in that the medicinal composition plays at a temperature of 23-37 DEG C Effect.